Stable associations of syntrophic fermentative organisms and populations that consume fermentation products play key roles in the anaerobic biodegradation of chlorinated organic contaminants. The involvement of these syntrophic populations is essential for mineralization of chlorinated aromatic compounds under methanogenic conditions. The fermentative production of low levels of hydrogen (H2) can also be used to selectively deliver a limiting electron donor to dehalogenating organisms and achieve complete dehalogenation of chlorinated aliphatic contaminants such as tetrachloroethene. Thus, tracking the abundance of syntrophically coupled populations should aid in the development and monitoring of sustainable bioremediation strategies. In this study, two complementary nucleic acid–based methods were used to identify and assess relative changes or differences in the abundance of potentially important populations in complex anaerobic microbial communities that mineralized chlorinated aromatic compounds. Population dynamics were related to the consumption and production of key metabolic substrates, intermediates, and products. Syntrophus-like populations were detected in 3-chlorobenzoate–degrading communities derived from sediment or sludge digesters. In the presence of H2-consuming populations, characterized Syntrophus species ferment benzoate, a central intermediate in the anaerobic metabolism of 3-chlorobenzoate and 2-chlorophenol. A DNA probe that targeted characterized Syntrophus species was developed and used to quantify rRNA extracted from the 3-chlorobenzoate– and 2-chlorophenol–degrading communities. The level of rRNA targeted by the Syntrophus-specific probe tracked with the formation of benzoate during metabolism of the parent compounds. Hybridizations with an Archaea-specific probe and/or measurement of methane production demonstrated that methanogens directly benefited from the influx of benzoate-derived electron donors, and the activities of Syntrophus-like and methanogenic populations in the contaminant-degrading communities were closely linked.
Phenanthrene-degrading bacteria were isolated from a 1-m2 intertidal sediment site in Boston Harbor. Samples were taken six times over 2 years. A total of 432 bacteria were isolated and characterized by biochemical testing. When clustered on the basis of phenotypic characteristics, the isolates could be separated into 68 groups at a similarity level of approximately 70%. Several groups (a total of 200 isolates) corresponded to well-characterized species belonging the genera Vibrio andPseudomonas. Only 51 of the 437 isolates (<11.7% of the total) hybridized to a DNA probe that encodes the upper pathway of naphthalene and phenanthrene degradation in Pseudomonas putida NCIB 9816. A cluster analysis indicated that the species composition of the phenanthrene-degrading community changed significantly from sampling date to sampling date. At one sampling time, 12 6-mm-diameter core subsamples were taken within the 1-m2 site to determine the spatial variability of the degrading communities. An analysis of molecular variance, performed with the phenotypic characteristics, indicated that only 6% of the variation occurred among the 12 subsamples, suggesting that the subsamples were almost identical in composition. We concluded that the communities of phenanthrene-degrading bacteria in the sediments are very diverse, that the community structure undergoes significant change with time but does not vary significantly on a spatial scale of centimeters, and that the predominant genes that encode phenanthrene degradation in the communities are not well-characterized.
The response of a complex methanogenic sediment community to 2-chlorophenol (2-CP) was evaluated by monitoring the concentrations of this model contaminant and important metabolic intermediates and products and by using rRNA-targeted probes to track several microbial populations. Key relationships between the evolving population structure, formation of metabolic intermediates, and contaminant mineralization were identified. The nature of these relationships was intrinsically linked to the metabolism of benzoate, an intermediate that transiently accumulated during the mineralization of 2-CP. Before the onset of benzoate fermentation, reductive dehalogenation of 2-CP competed with methanogenesis for endogenous reducing equivalents. This suppressed H 2 levels, methane production, and archaeal small-subunit (SSU)-rRNA concentrations in the sediment community. The concentrations of bacterial SSU rRNA, including SSU rRNA derived from "Desulfovibrionaceae" populations, tracked with 2-CP levels, presumably reflecting changes in the activity of dehalogenating organisms. After the onset of benzoate fermentation, the abundance of Syntrophuslike SSU rRNA increased, presumably because these syntrophic organisms fermented benzoate to methanogenic substrates. Consequently, although the parent substrate 2-CP served as an electron acceptor, cleavage of its aromatic nucleus also influenced the sediment community by releasing the electron donors H 2 and acetate. Increased methane production and archaeal SSU-rRNA levels, which tracked with the Syntrophus-like SSUrRNA concentrations, revealed that methanogenic populations in particular benefited from the input of reducing equivalents derived from 2-CP.
Lengthy adaptation periods in laboratory studies evaluating the potential for contaminant biodegradation in natural or engineered environments may indicate that the native microbial communities are not metabolizing the contaminants in situ. In this study, we characterized the adaptation period preceding the biodegradation of 3-chlorobenzoate in anaerobic communities derived from lake sediment and wastewater sludge digesters. The importance of alternative mechanisms of adaptation of the anaerobic communities to 3-chlorobenzoate was evaluated by monitoring the concentrations of metabolic substrates and products as well as the levels of total small subunit (SSU) rRNA and SSU rRNA from populations thought to be important in 3-chlorobenzoate mineralization. The anaerobic environments from which the 3-chlorobenzoate-degrading communities were derived contained different levels of endogenous substrates. Increasing methane levels in the digester and sediment communities and decreasing chemical oxygen demand concentrations in the sediment community during the adaptation periods revealed that endogenous substrates were preferentially utilized relative to 3-chlorobenzoate. Methane and chemical oxygen demand concentrations leveled off concomitantly with the onset of 3-chlorobenzoate biodegradation, suggesting that depletion of the preferentially degraded endogenous substrates stimulated 3-chlorobenzoate metabolism. Consistent with these observations, adaptation to 3-chlorobenzoate occurred more rapidly in digester samples that were depleted of endogenous substrates compared to samples that contained high levels of these biodegradable compounds. Other potential adaptation mechanisms, e.g., genetic change or selective population enrichment, appeared to be less important based on the reproducibility and relative lengths of the adaptation events, trends in the SSU rRNA levels, and/or amplification of SSU rRNA genes from key populations.
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